Adsorptive resin for microorganisms

ABSTRACT

The present invention relates to absorption resins comprising vinyl copolymers which do not dissolve in water and are capable of absorbing microorganisms without inactivating the microorganisms and also adsorbents with a large contact area which are capable of absorbing microorganisms efficiently as well as methods of making the resins and the adsorbents.

This is a continuation of copending application Ser. No. 07/550,967filed on Jul. 11, 1990 now abandoned.

DETAILED EXPLANATION OF THE INVENTION

1. Industrial Field of Application

The present invention relates to absorptive resins which do not dissolvein water and are capable of adsorbing microorganisms withoutinactivating the microorganisms and also adsorbents with a large contactarea which are capable of adsorbing microorganisms efficiently as wellas methods of making the resins and the adsorbents.

2. Prior Art

It has previously been proposed to provide means to adsorbmicroorganisms in service water or in sewage by using water solublechemical substances such as chlorine which posses a germicidal power.However, those chemical substances, in general, are often toxic andtherefore unsuitable for use in drinking water or water for productionof foods as well as medicines. Chlorine, for instance, produces organichalogens such as trihalomethane during water treatment. Trihalomethanewas recently found to be carcinogenic and also subject to biologicalconcentrations, a rising problem of environmental protection movements.

There has long been a need for a new substance which is water insolubleand able to remove microorganisms.

In the field of bioreactors and biosensors, enzyme produced bymicroorganisms which are fixed to carriers live are being considered.Some methods such as physical adsorption and covalent bonding arepresently known to be capable to fix microorganisms to carriers. Thephysical adsorption provides weak cohesion and microorganisms may driftaway. On the other hand, the covalent bonding provides relatively strongcohesion, but enzyme may denature in reaction or die out. The demandsfor a carrier which is capable of stably fixing microorganisms withoutexterminating or inactivating microorganisms have been strong.

Japanese patent publication No. 41641/87 proposes insoluble highpolymers comprising cross-linked polyvinyl pyridinium halide as amaterial to satisfy the demands for a remover of microorganisms in watertreatment as well as for a carrier in a bioreactor.

This chemical compound, insoluble in water as well as capable ofefficiently adsorbing and retaining microorganisms live, makes anexcellent adsorbent of microorganisms and does not contaminate theenvironment.

Said cross-linked polyvinyl pyridinium halide, however, is insoluble notonly in water but also in an ordinary organic solvent. A number ofproblems such as working on the chemical is difficult for it is usuallyused only in a solid state arise as beads and the surface area per unitweight is small, decreasing adsorption of microorganisms. In order toincrease surface area, the size of beads, for example, could be madesmaller, however, such tiny beads might cause clogging of the system.

In view of the present situation described above, the present inventionis to overcome the problems of known adsorptive resins that areinsoluble not only in water but also in a organic solvent difficult toworking with and poor adsorptive performance for the weight.

Accordingly it is an object of the present invention to provideadsorptive resins which are insoluble or hard to dissolve in water butsoluble in an organic solvent, easy to work on, capable of efficientlyadsorbing microorganisms by having a large surface area per unit weightand which do not inactivate the microorganisms and also to provideadsorbents which comprise the adsorptive resins, as well as methods tomake the adsorptive resins and the adsorbents.

SUMMARY OF THE INVENTION

An adsorptive resin of the present invention comprises vinyl copolymersof the formula ##STR1## wherein R¹ is a benzyl group, an alkyl group of4 to 16 carbon atoms or a pentafluorophenylmethyl group, R² is ahydrogen atom or an alkyl group of 1 to 3 carbon atoms, X is a halogenatom, and Y is a hydrogen atom, an alkyl group of 1 to 3 carbon atoms, abenzyl group, and ether group, a carboxyl group, a carboxylic acid estergroup or an aryl group.

An adsorptive resin of the present invention possesses an adsorptivepower comparable to cross-linked polyvinyl pyridinium halide, and isinsoluble or hard to dissolve in water but soluble in an organic solventas well as capable of making an organic solution. Accordinglyimpregnation of the adsorptive resin into or coating it onto a basematerial has become possible, which was impossible with the knowncross-linked polyvinyl pyridinium halide.

DETAILED DESCRIPTION OF THE INVENTION

An adsorptive resin of the present invention comprises vinyl copolymersof the formula, ##STR2## wherein R¹ is a benzyl group, an alkyl group of4 to 16 carbon atoms or a pentafluorophenylmethyl group, R² is ahydrogen atom or an alkyl group of 1 to 3 carbon atoms, X is a halogenatom, and Y is a hydrogen atom, an alkyl group of 1 to 3 carbon atoms, abenzyl group, an ether group, a carboxyl group, a carboxylic acid estergroup or an aryl group,

which are obtained after copolymerizing 4-vinyl pyridine and monovinylmonomer, and acting halide,

said vinyl copolymers being random copolymers or block copolymers.

Monovinyl monomers to be used for copolymerization are olefine such asethylene, propylene and butylen and the like, styrene, vinyl acetate,acrylic acid, acrylic acid ester, methacrylic acid, methacrylic acidester, aliphatic vinyl ester, acrylnitrile and their derivatives. Othersmay also be used alone or in combination. However, monovinyl monomersthat posses a highly hydrophilic functional group should be avoided, forsome copolymers obtained using such hydrophilic functional group maybecome water soluble.

The ratio of 4-vinyl pyridine and monovinyl monomer (n:m) differsaccording to the kinds or copolymerizational degrees. It is recommendedthat n:m be within an approximate range of 10:90 to 90:10. If theproportion of 4-vinyl pyridine is below the range, a sufficientadsorptive power cannot be gained and if it exceeds the range, theresulting copolymers become highly water soluble.

The copolymerizational degree of said vinyl copolymers should be atleast 300. If less than that degree, the obtained copolymers becomehighly water soluble.

The copolymers of 4-vinyl pyridine and monovinyl monomer form afunctional group of the formula,

    .[.X.sup.- --R.sub.1 --N.sup.-.].

    .Iadd.X.sup.- R.sub.1 --N .Iaddend.

by quaternizing pyridin with halide such as alkyl halide, benzyl halideand pentafluorophenylmethyl halide.

This functional group is considered to be a significant in adsorbingmicroorganisms and retaining the microorganisms in an active state. Themechanism of it is not yet clear, however, it is estimated that anelectrostatical interaction is a key factor for the mechanism. That is,the electrically plus charged functional group attracts microorganismsthe surface of whose cell is generally minus charged.

The term "microorganisms" is used herein to mean bacteria, fungi, algae,viruses and the like.

An excellent remover of microorganisms for water treatment and anexcellent adsorbent of microorganisms for use as a carrier in abioreactor or a biosensor can be obtained by firmly adhering to thesurface of a base material an adsorptive resin comprising vinylcopolymers in the adhering steps as described below.

An adsorptive resin of microorganisms produced in the process describedin the above is soluble in an organic solvent but insoluble or hardlysoluble in water.

An adsorbent of microorganisms is obtained by:

dissolving an adsorptive resin of the present invention in an organicsolvent such as alcohol, ester and ether, making a non-water solution;

adhering the non-water solution on a base material by impregnating,spraying or coating; and

drying the base material so as to firmly adhere the adsorptive resin tothe surface of the base material.

From a standpoint of easy handling, a desirable organic solvent isalcohol. And, a desirable way to adhere an adsorptive resin to a basematerial as widely inside and evenly as possible is impregnation.

There exists no particular limitation to the amount of said adsorptiveresin of microorganisms to be used on a base material, however, itprovides a peak adsorption and cost performance in the range between0.001 and 1 μm in thickness of adhered adsorptive resin, which meansroughly 0.001 to 100 wt % of a base material. When it is less than 0.001wt %, a sufficient enough adsorptive power cannot be expected and whenit exceeds 100 wt , an increase of the amount of an adsorptive resindoes not proportionally result in an increase of the adsorptive power.When a nonwoven fabric is used as a base material, the range of 0.01 to20 wt % provides a high adsorption and fine cost performance.

A variety of materials such as a porous material like a nonwoven fabric,a woven fabric, knitted goods, paper, foamy materials and a ceramicsinter, organic or inorganic beads, or a honeycomb or a multistage boardcan be used as a base material for the adsorbent of microorganisms, andit is possible to select from among either single or compound, organic,inorganic or metal substances. Among these, porous materials are bestrecommended as they have a large surface area. And among porousmaterials, a nonwoven fabric is better selected for it isthree-dimensional, gives a large surface area and is able to lowerpressure loss. In addition, an adsorptive resin evenly and thinlyadheres to the surface of fibers that constitute a nonwoven fabric, thesurface area per weight of an adsorptive resin becoming much larger thanthat in the case of beads. It is then possible to sharply increase thesurface area of a nonwoven base material that contacts microorganisms inwater treatment and accordingly the efficiency of reaction in abioreactor. It is also possible, by controlling the density of anonwoven fabric, to give the nonwoven base material a structure which ishard to clog when a large quantity of microorganisms is adsorbed.

When an adsorbent of microorganisms is used in water treatment, water ispurified by removal of microorganisms in the water by the adsorbent.When it is used for a bioreactor either in an atmosphere or in a liquid,it adsorbs microorganisms possessing specific functions in a living anda highly active state, and some products can be obtained by activatingenzyme in the microorganisms, or substances can be sorted.

Further, when an adsorbent of microorganisms is used for an airfilter ora mask, viruses and bacteria can be collected together with otherharmful substances and dusts in the air.

It is also possible to adhere an adsorptive resin of the presentinvention to the surface of a base material together with a germicide.

The term "germicide" as used herein includes a germicide that sterilizesmicroorganisms and a germicide that prevents microorganisms frompropagating or holds microorganisms under control. Suitable germicidesare antibiotics such as polymixin, positive surfactants such asquaternary ammonium salt, ampholytic surfactants such asalkylaminoethylglycine, biguanides such as chlorhexidine andpolyhexamethylene biguanide, higher fatty acids such as undecylenicacid, metal or metal ion and phenol.

These germicides can be adhered to the surface of a base materialtogether with an adsorptive resin of the present invention byimpregnating, spraying or coating. Also, when a base material composinga porous material such as a nonwoven fabric is used, these germicidescan be placed in the base material in the form of germicidal fibers orresistive fibers that contain a germicide.

Germicides used for an absorbent of microorganisms in an atmosphere canbe any of those above, however, when the adsorbent is used in water,especially in treatment of drinking water, it is harmful if germicidesare dissolved in the water. In order to avoid the problem, germicides tobe used in water should be insoluble or hardly soluble in water.Recommendable germicides are metal powders such as gold, silver orcopper powders, antibacterial zeolite made by permuting zeolite withgermicidal metal ions, polymer- type fixed germicides made by fixingbiguanide or quaternary ammonium salt to polymers such as polyvinyl,polyacrylate, polyester and polyamide or silicon-type fixed germicidessuch as 3-(trimetoxysilil)-propyltrimethyloctadecil ammoniumchrolide.

The adhesion process of said germicides to a base material can beperformed at any point of the procedure. It can be performed beforeadhering an adsorptive resin of microorganisms of the present inventionto the base material, after, or simultaneous with the adhesion of theresin. The method of adhering a germicide to a base material may bedirectly adhering a germicide to a base material or impregnating,spraying or coating an organic solvent solution in which a germicide isdissolved or scattered, then blowing away the organic solvent.Especially when a germicide is to be adhered together with an adsorptiveresin of the present invention to a base material, it is recommendedthat the germicide be dissolved or scattered in an organic solventsolution of the adsorptive resin.

In order to adhere a germicide in a base material, the germicide iscontained in the component of a base material such as a fiber, a porousmaterial, or a film and a resin sheet or a germicidal functional groupis induced into the component.

The amount of a germicide will depend on the kind and the germicidalpower of the germicide, however, the amount should be enough to preventcollected microorganisms from propergating.

An adsorbent of the present invention is capable of adsorbingmicroorganisms in water or in the air very effectively, and eithersterilizes the microorganisms with a germicide or prevents them frompropagating.

According to the research reports made by the inventors and otherresearchers of the present invention, it is clear that the adsorptivecapability of an adsorbent of microorganisms of the present invention isexcellent in water but not very satisfactory in the air. Therefore, whenan adsorbent of microorganisms is to be used in an atmosphere, it isdesired that the atmosphere is a highly moist gas or a moistener existson the surface of the base material together with an adsorptive resin ofthe present invention. It is better to utilize a humidifying means suchas a humidifier to humidify the atmosphere.

Moisteners to be used for said adsorbents of microorganisms of thepresent invention can be any that adsorbs moisture in an atmosphere, forexample, alkali earth metallic salts such as calcium chloride andmagnesium chloride, deliquescent substances such as lithium chloride,potassium metasolicic acid and titanium sulfate, water soluble polymerssuch as polyvinyl alcohol, polyacrylate, polyvinyl pyrrolidone andcarboxymethylcellulose, silica gel, zeolite, collagen, ethylene glycol,diphosphoric pentoxide, magnesium oxide or calcium oxide.

These moisteners can be adhered to the surface of the base materialtogether with said adsorptive resin by impregnating, spraying orcoating.

Adhesion of a moistener is to be conducted before or after adhesion ofan adsorptive resin consisting of said vinyl copolymers, or conductedsimultaneously. Especially when adhesion of a moistener is conductedsimultaneously with adhesion of an adsorptive resin a moistener is to beput in an organic solvent solution of said adsorptive resin ofmicroorganisms to coexist. If a moistener is soluble in an organicsolvent, it dissolves in the solution together with an adsorptive resin,and if it is not soluble, it is scattered in the solution.

The amount of a moistener is recommended to be approximately 5-500 wt %of the amount of an adsorptive resin to be used though, the amountdepends on the kind of a moistener and the adsorptive power of moisture.When the amount is less than the range, it cannot supply enough moistureto keep active the adsorptive part of an adsorptive resin of the presentinvention, and when the amount is in excess of the range, the adsorptivepower of moisture does not improve in proportion to the increase theamount of the moistener.

An adsorbent of microorganisms using said adsorptive resin ofmicroorganisms can be used for a mask or an airfilter. In these cases,an adsorbent may be used without modification, or may be used with agermicide and a moistener on the surface of a base material togetherwith an adsorptive resin of the present invention. In order to adhere agermicide together with a moistener, a germicide is to be adhered to thesurface of a base material or a germicidal fiber containing a germicideor a germicidal fiber to which a functional group is induced is to beused as a component of a base material. When a germicide is usedtogether with an adsorptive resin of the present invention, the adheredmicroorganisms become unable to propagate, which prevents clogging ofthe system and elongates the life of a mask and an airfilter.

WORKING EXAMPLES

Adsorptive resins and adsorbents of microorganisms of the presentinvention, and the methods of making them are described in detail in thefollowing, with the help of working examples.

4-vinyl pyridine, stylene and methyl methacrylate monomer for thefollowing working examples are all obtainable by refining chemicals onthe market by a process such as decompression distillation.

WORKING EXAMPLE 1

An adsorptive resin comprising vinyl copolymers was obtained in thesteps:

putting in a reactor 0.84 g (5 m mol) of 2,2'-azobisisobutyronitrile(purity 98%) and 533.84 g of ethanol (purity 99%) and regulating acatalytic solution;

putting in 4-vinylpyridine (105.14 g, 1,000 m mol) and stylene (156.23g, 1,500 m mol) in the proportion of 1:1.5 mol; and

copolymerizing them in an nitrogen atmosphere for six hours at 78° C.;and then

putting in 174.53 g (1,000 m mol) of benzylbromide (purity 98%) andquaternizing them for six hours at 78° C.

Table 1 shows the description above in figures.

For the convenience of easy understanding and easy calculation, theamount of 4-vinyl pyridine is uniformly made one mol.

                  TABLE 1                                                         ______________________________________                                                                         2,2'-                                                                         azobisiso-                                   chemical 4-vinyl          benzyl buty-   ethyl                                compound pyridine stylene bromide                                                                              ronitrile                                                                             alcohol                              ______________________________________                                        abbreviation                                                                           4VP      ST      BzBr   AIBN    EtOH                                 ratio of 1        1.5                                                         monomer                                                                       mol %    40       60      40     0.2                                          (monomer =                                                                    100)                                                                          molecular                                                                              105.14   104.15  171.04 164.21  46.07                                weight                                                                        (g/mol)                                                                       purity   100      100     98     98      99                                   (%)                                                                           calculative                                                                            1,000    1,500   1,000  5                                            annexing                                                                      (m mol)                                                                       annexing 105.14   156.23  174.53 0.84    *533.84                              amount                                                                        (g)                                                                           ______________________________________                                         *In order to make monomer concentration 45 wt % the necessary EtOH =          {[(105.14 - 156.23 - 171.04)/0.45] - (105.14 - 156.23 - 171.04)}/0.99 =       533.84                                                                   

On the other hand, a fiber web made of rayon fiber (1.5 deniers) wasmade into a nonwoven fabric of 90 g/m² and 0.5 mm thick by waterentanglement.

After impregnating an 0.5 wt % ethanol solution in which said vinylcopolymers were dissolved into said nonwoven fabric, the nonwoven fabricwas dried at 75° C. and an adsorbent of microorganisms of which 1.5 wt %was copolymers was obtained.

Stamping out discs whose diameter was 3 cm from the adsorbent, andfilling 30 discs in a column whose inside diameter was 3 cm. asuspension with Escherichia coli concentration of 46×10⁸ /ml in 0.85%salt water was sent through the column at a velocity of 45 ml/hr.

Table 3 shows the results of removal rates of living microorganisms inthe filtrate, which was taken at regular intervals of time by the agarplate culture method.

COMPARISON EXAMPLE 1

Another nonwoven fabric was made in the same process of Working Example1, except that no adsorptive resin was used, and 30 discs were filled inanother column in the same manner, and a suspension with an Escherichiacoli concentration of 5.5×10⁸ /ml in 0.85% salt water was sent throughthe column at a velocity of 35 ml/hr.

Table 3 shows the results of removal rate of living microorganism in thefiltrate, which was taken at regular intervals of time by the agar plateculture method.

WORKING EXAMPLE 2

An adsorptive resin was obtained as described in Working Example 1,except that the amount of ethanol was changed to 47.96 g, and 127.87 g(1,000 m mol) of benzyl chloride (purity 99%) was used instead of benzylbromide.

Table 2 shows the description above in figures.

For the convenience of easy understanding and easy calculation, theamount of 4-vinyl pyridine is uniformly made one mol.

Discs taken from the absorbent made in the same process of WorkingExample 1 were filled in still another column in the same manner as inWorking Example 1 and a suspension with Escherichia coli concentrationof 5.6×10⁸ /ml in 0.85% salt water was sent through the column at avelocity of 40 ml/hr.

Table 3 shows the results of removal rate of living microorganisms inthe filtrate, which was taken at regular intervals of time by the agarculture method.

As is apparent from Table 3, the adsorbents of microorganisms of WorkingExamples 1 and 2, to both of which vinyl copolymers were adhered, showsan excellent adsorption of microorganisms and a high removal rate ofmicroorganisms, while the adsorbent without vinyl copolymers inComparison Example 1 shows poor adsorption, which went poorer with time,the removal rate of microorganism becoming mere 10% or so in 2 hours.

                  TABLE 1                                                         ______________________________________                                                                         2,2'-                                                                         azobisiso-                                   chemical 4-vinyl          benzyl buty-   ethyl                                compound pyridine stylene bromide                                                                              ronitrile                                                                             alcohol                              ______________________________________                                        abbreviation                                                                           4VP      ST      BzCl   AIBN    EtOH                                 ratio of 1        1.5                                                         monomer                                                                       mol %    40       60      40     0.2                                          (monomer =                                                                    100)                                                                          molecular                                                                              105.14   104.15  126.59 164.21  46.07                                weight                                                                        (g/mol)                                                                       purity   100      100     98     98      99                                   (%)                                                                           calculative                                                                            1,000    1,500   1,000  5                                            annexing                                                                      (m mol)                                                                       annexing 105.14   156.23  127.87 0.84    478.96                               amount                                                                        (g)                                                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                                    Working Working   Comparison                                                  Example 1                                                                             Example 2 Example 1                                       ______________________________________                                        vinyl copolymers                                                                            existent  existent  none                                        quaternizing  benzyl    benzyl                                                component     bromide   chloride                                              microorganisms                                                                              4.6 × 10.sup.8                                                                    5.6 × 10.sup.8                                                                    5.5 × 10.sup.8                        in water (No./ml)                                                             microorganisms                                                                              1.4 × 10.sup.4                                                                    4.6 × 10.sup.4                                                                    3.1 × 10.sup.8                        in filtrated water                                                            1 hr. later (No./ml)                                                          rate of removal (%)                                                                         99.997    99.992    43.6                                        microorganism 3.7 × 10.sup.5                                                                    5.5 × 10.sup.5                                                                    4.9 × 10.sup.8                        in filtrated water                                                            2 hrs. later (No./ml)                                                         rate of removal (%)                                                                         99.92     99.90     10.9                                        ______________________________________                                    

WORKING EXAMPLE 3

Discs taken from yet another adsorbent made in the same process ofWorking Example 1 were filled in yet another column in the same manneras in Working Example 1, and a suspension with Staphylococcus aureus;concentration of 2.8×10⁸ /ml in 0.85% salt water was sent through thecolumn at a velocity of 60 ml/hr.

Table 4 shows the results of removal rate of living microorganisms inthe filtrate, which was taken at regular intervals of time.

WORKING EXAMPLE 4

Discs taken from yet still another adsorbent made in the same process ofWorking Example 1 were filled in yet still another column in the samemanner as in Working Example 1, and a suspension with Pseudomonasaeruginosa concentration of 6.2×10⁸ /ml in 0.85% salt water was sentthrough the column at a velocity of 30 ml/hr.

Table 4 shows the results of removal rate of living microorganism in thefiltrate, which was taken at regular intervals of time.

As is apparent from Table 4, adsorbents of microorganisms with the useof an adsorptive resin of the present invention showed an excellentadsorption disregarding types of microorganisms.

                  TABLE 4                                                         ______________________________________                                                    Working Working   Working                                                     Example 1                                                                             Example 3 Example 4                                       ______________________________________                                        type of       Escherichia                                                                             staphylo- Pseudomonas                                 microorganism coli      coccus    Aeruginosa                                                          Aureus                                                microorganism 4.6 × 10.sup.8                                                                    2.8 × 10.sup.8                                                                    6.2 × 10.sup.8                        in water (No./ml)                                                             microorganism 1.4 × 10.sup.4                                                                    9.2 × 10.sup.3                                                                    8.2 × 10.sup.4                        in filtrated water                                                            1 hr. later (No./ml)                                                          rate of removal (%)                                                                         99.997    99.990    99.990                                      microorganism 3.7 × 10.sup.5                                                                    5.2 × 10.sup.5                                                                    2.2 × 10.sup.8                        in filtrated water                                                            2 hrs. later (No./ml)                                                         rate of removal (%)                                                                         99.92     99.81     99.64                                       ______________________________________                                    

WORKING EXAMPLE 5

Twenty discs with a diameter of 1.6 cm taken from another adsorbent madein the same process of Working Example 1 were filled in another columnwith an inside diameter of 1.6 cm in the same manner as in WorkingExample 1, and a suspension with T-4 bacteriophage concentration of1.3×10⁶ /ml in 0.85% salt water was sent through the column at avelocity of 20 ml/hr.

Table 5 shows the results of removal rate of living microorganisms inthe filtrate, which was taken at regular intervals of time.

COMPARISON EXAMPLE 2

Without using an adsorptive resin of the present invention, a nonwovenfabric was filled in a column, and a suspension with T-4 bacteriophageconcentration of 1.3×10⁶ /ml in 0.85% salt water was sent through thecolumn at a velocity of 20 ml/hr.

Table 5 shows the results of removal rate of living microorganisms inthe filtrate, which was taken at regular intervals of time.

Table 5 also shows that an absorbent with the use of an adsorptive resinworks effectively on viruses which are tinier than fungi.

                  TABLE 5                                                         ______________________________________                                                    Working    Comparison                                                         Example 5  Example 2                                              ______________________________________                                        vinyl copolymers                                                                            existent     none                                               type of       T-4          T-4                                                microorganisms                                                                              bacteriophage                                                                              bacteriophage                                      microorganisms                                                                              1.3 × 10.sup.6                                                                       1.3 × 10.sup.6                               in water                                                                      (No./ml)                                                                      microorganism 2.3 × 10.sup.2                                                                       4.6 × 10.sup.5                               in filtrated water                                                            1 hr. later                                                                   (No./ml)                                                                      rate of removal                                                                             99.98        65.0                                               (%)                                                                           ______________________________________                                    

WORKING EXAMPLE 6

An adsorbent of microorganisms was obtained as described in WorkingExample 1, except that the ratio of 4-vinyl pyridine and stylene waschanged to 1:3 mol, that is the amounts of 4-vinyl pyridine, stylene,2,2'-azobisisobutyronitrile and ethanol are 105.14 g 312.45 g (3,000 mmol), 1.34 g (8 m mol) and 726.70 g respectively

Table 6 shows the description above in figures.

For the convenience of easy understanding and easy calculation, theamount of 4-vinyl pyridine is uniformly made one mol.

Discs taken from another adsorbent made in the same process of WorkingExample 1 were filled in another column in the same manner as in WorkingExample 1, and a suspension with Escherichia coli concentration of2.3×10⁸ /ml of 0.85% salt water was sent through the column at avelocity of 70 ml/hr.

Table 7 shows the results of removal rate of living microorganisms inthe filtrate, which was taken at regular intervals of time.

WORKING EXAMPLE 7

An absorbent of microorganisms was obtained as described in WorkingExample 1, except that methyl methacrylate was used instead of styleneand the ratio of 4-vinyl pyridine and stylene was 1:1.5 mol, that is theamounts of methyl methacrylate and ethanol are 150.18 g (1,500 m mol)and 526.37 g respectively.

Table 8 shows the description above in figures.

For the convenience of easy understanding and easy calculation, theamount of 4-vinyl pyridine is uniformly made one mol.

As in Working Example 1, a suspension with Escherichia coliconcentration of 3.8×10⁸ /ml in 0.85% salt water was sent through acolumn at a velocity of 50 ml/hr, and the rate of removal was taken.Table 7 shows the results.

Table 7 shows that the proportions of 4-vinyl pyridine and vinylmonomer, which are raw material for vinyl copolymers of the presentinvention, affect adsorption of microorganisms.

Table 7 also shows that even when a component of vinyl monomers ischanged, an adsorbent of microorganisms with satisfactory adsorptiveefficiency can be obtained.

                  TABLE 6                                                         ______________________________________                                                                         2,2'-                                                                         azobisiso-                                   chemical 4-vinyl          benzyl buty-   ethyl                                compound pyridine stylene bromide                                                                              ronitrile                                                                             alcohol                              ______________________________________                                        abbreviation                                                                           4VP      ST      BzBr   AIBN    EtOH                                 ratio of 1        3                                                           monomer                                                                       mol %    25       75      25     0.2                                          (monomer =                                                                    100)                                                                          molecular                                                                              105.14   104.15  171.04 164.21  46.07                                weight                                                                        (g/mol)                                                                       purity   100      100     98     98      99                                   (%)                                                                           calculative                                                                            1,000    3,000   1,000  8                                            annexing                                                                      (m mol)                                                                       annexing 105.14   312.45  174.53 1.34    726.70                               amount                                                                        (g)                                                                           ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                                 Working  Working    Working                                                   Example 1                                                                              Example 6  Example 7                                        ______________________________________                                        vinyl      styrene    styrene    methyl                                       monomer                          methacrylate                                 4-vinyl-   40/60      25/75      40/60                                        pyridine/                                                                     vinyl monomer                                                                 micro-     4.6 × 10.sup.8                                                                     2.3 × 10.sup.8                                                                     3.8 × 10.sup.8                         organisms                                                                     in water                                                                      (No./ml)                                                                      micro-     1.4 × 10.sup.4                                                                     1.4 × 10.sup.6                                                                     8.0 × 10.sup.3                         organisms                                                                     in filtrated                                                                  water 1 hr.                                                                   later (No./ml)                                                                rate of    99.997     99.4       99.998                                       removal (%)                                                                   micro-     3.7 × 10.sup.5                                                                     6.4 × 10.sup.6                                                                     6.1 × 10.sup.5                         organisms                                                                     in filtrated                                                                  water 2 hrs.                                                                  later (No./ml)                                                                rate of    99.92      97.2       99.84                                        removal (%)                                                                   ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                                                         2,2'-                                                          methyl         azobisiso-                                   chemical 4-vinyl  metha-  benzyl buty-   ethyl                                compound pyridine crylate bromide                                                                              ronitrile                                                                             alcohol                              ______________________________________                                        abbreviation                                                                           4VP      MMA     BzBr   AIBN    EtOH                                 ratio of 1        1.5                                                         monomer                                                                       mol %    40       60      40     0.2                                          (monomer =                                                                    100)                                                                          molecular                                                                              105.14   100.12  171.04 164.21  46.07                                weight                                                                        (g/mol)                                                                       purity   100      100     98     98      99                                   (%)                                                                           calculative                                                                            1,000    1,500   1,000  5                                            annexing                                                                      (m mol)                                                                       annexing 105.14   150.18  174.53 0.84    526.37                               amount                                                                        (g)                                                                           ______________________________________                                    

WORKING EXAMPLE 8

An adsorbent of microorganisms was obtained in the same process as inWorking Example 1, except that a porous base material was made ofneedlepunch nonwoven fabric of 126 g/m² and 0.87 mm in thickness, whichis made of polyester fiber (1.5 deniers).

Stamping out discs whose diameter was 3 cm from the absorbent, andfilling another column, whose inside diameter was 3 cm, with 23 of thediscs, a suspension with Escherichia coli concentration of 5.5×10⁸ /mlin 0.85% salt water was sent through the column at a velocity of 35ml/hr.

Table 9 shows the results of removal rate of microorganisms in thefiltrate, which was taken at regular intervals of time.

WORKING EXAMPLE 9

An adsorbent of microorganism was obtained in the same process as inWorking Example 1, except that a porous base material was made ofneedlepunch nonwoven fabric of 151 g/m² and 1.11 mm in thickness, whichis made of acrylic fiber (1.3 deniers).

Stamping out discs whose diameter was 3 cm from the absorbent, andfilling a column whose inside diameter was 3 cm with 18 of the discs, asuspension with Escherichia coli concentration of 3.9×10⁸ /ml in 0.85%salt water was sent through the column at a velocity of 50 ml/hr.

Table 9 shows the results of removal rate of microorganisms in thefiltrate, which was taken at regular intervals of time.

WORKING EXAMPLE 10

An adsorbent of microorganisms was obtained in the same process as inWorking Example 1, except that a porous base material was made ofwet-type nonwoven fabric of 150 g/m² and 1.05 mm in thickness, which ismade of glass fiber (6 μm).

Stamping out discs whose diameter was 3 cm from the adsorbent, andfilling another column whose inside diameter was 3 cm with 24 of thediscs, a suspension with Escherichia coli concentration of 2.8×10⁸ /mlin 0.85% salt water was sent through the column at a velocity of 55ml/hr.

Table 9 shows the results of removal rate of microorganisms in thefiltrate, which was taken at regular intervals of time.

WORKING EXAMPLE 11

An adsorbent of microorganisms was obtained in the same process as inWorking Example, 1 except that a porous base material was made ofneedlepunch nonwoven fabric of 135 g/m² and 0.90 mm in thickness, whichis made of rayon fiber (1.5 deniers).

Stamping out discs whose diameter was 3 cm from the adsorbent, andfilling a column whose inside diameter was 3 cm with 26 of the discs, asuspension with Escherichia coli concentration of 3.3×10⁸ /ml in 0.85%salt water was sent through the column at a velocity of 55 ml/hr.

Table 9 shows the results of removal rate of microorganisms in thefiltrate, which was taken at regular intervals of time.

Table 9 also shows that the type of porous material affects adsorptionof an adsorbent of microorganisms a little, but all adsorbents stillretain an adsorptive power.

                  TABLE 9                                                         ______________________________________                                               Working Working   Working   Working                                           Example 8                                                                             Example 9 Example 10                                                                              Example 11                                 ______________________________________                                        porous   NP        NP        wet-type                                                                              NP                                       material nonwoven  nonwoven  nonwoven                                                                              nonwoven                                          fabric    fabric    fabric  fabric                                   made of  polyester acrylic   glass   rayon                                    micro-   5.5 × 10.sup.8                                                                    3.9 × 10.sup.8                                                                    2.8 × 10.sup.8                                                                  3.3 × 10.sup.8                     organisms                                                                     in water                                                                      (No./ml)                                                                      micro-   5.5 × 10.sup.8                                                                    7.8 × 10.sup.4                                                                    5.6 × 10.sup.5                                                                  2.3 × 10.sup.5                     organisms                                                                     in filtrated                                                                  water                                                                         1 hr. later                                                                   (No./ml)                                                                      remove rate                                                                            99.0      99.98     99.8    99.93                                    (%)                                                                           micro-   2.5 × 10.sup.7                                                                    1.2 × 10.sup.6                                                                    4.2 × 10.sup.6                                                                  2.0 × 10.sup.6                     organisms                                                                     in filtrated                                                                  water                                                                         2 hrs. later                                                                  (No./ml)                                                                      removal rate                                                                           95.5      99.7      98.5    99.4                                     (%)                                                                           ______________________________________                                    

WORKING EXAMPLE 12

An adsorptive resin was obtained as described in Working Example 6.

Table 6 shows the contents of the above operation.

A nonwoven fabric (90 g/m², 0.5 mm in thickness) was obtained by waterentangling a fiber web made of rayon fiber (1.5 deniers)

An adsorbent of microorganisms, the proportion of copolymers against thenonwoven fabric of which was 1.5 wt %, was obtained in the steps:

dissolving said adsorptive resin of microorganisms into an ethanolsolution;

also impregnating the ethanol solution, in which germicidal zeolitepowder (Kanebo's Bactekiller) is scattered the weight ratio of vinylcopolymers and germicidal zeolite powder being 97.3, into said nonwovenfabric; and

drying at 75° C.

Said absorbent of microorganisms (5 cm×50 cm) made into a spiral shapewas put in a container and a 200 ml suspension of 0.85% sterile saltwater in which colon bacilli were suspended (1×10² /ml) was also put inthe container. The suspension was stirred with a magnetic stirrer. Twohours later, the number of live colon bacilli in the liquid was measuredby the agar plate culture method, and the measurement 0/ml was obtained,showing the 2×10⁴ (1×10² /ml×200 ml) of colon bacilli were adsorbed.

Next, the adsorbent of microorganisms which had adsorbed colon bacilliwas taken out, cut into 5 cm×5 cm pieces and were placed on agar culturemedia, cultured for 48 hours at 32° C. and the number of colonies wascounted. These were two colonies of colon bacilli on an agar culturemedium.

The result shows that the adsorbent of Working Example 12 is excellentin adsorption of colon bacilli and possesses a function to kill colonbacilli. Therefore, it is apparent that the adsorbent of Working Example12 is capable of effectively removing microorganisms in water withoutclogging the system.

WORKING EXAMPLE 13

An adsorptive resin was obtained as described in Working Example 1,except that the ratio of 4-vinyl pyridine and stylene was changed to1:2.5 mol, that is the amounts of 4-vinyl pyridine, stylene,2,2'-azobisisobutyronitrile and ethanol are 105.14 g, 260.38 g (2,500 mmol), 1.17 g (7 m mol) and 662.42 g respectively.

Table 10 shows the description above in figures.

An adsorbent of microorganisms (adsorptive resin is 1.5 wt % of nonwovenfabric) was obtained by impregnating an ethanol solution of saidadsorptive resin, powdered antibacterial zeolite and lithium chloride inthe proportion of 100:3:150 into a nonwoven fabric and drying at 75° C.

Said adsorbent (10 cm×120 cm) was pleated and made into a filter unit of10 cm×10 cm. A spray box of colon bacilli was installed on theupperstream side of the filter unit and a suction pump with a membranousfilter of 0.45 μm was installed on the downstream side.

After having moisturized the adsorbent well by sending through sterilemoist air for 30 minutes, a diluted isotonic sodium chloride solutioncontaining colon bacilli was sprayed and an atmosphere containing colonbacilli was sucked by the pump at a speed of 10 l/min. for 2 minutes.

After that operation, the filter unit made of an adsorbent ofmicroorganisms which had adhered colon bacilli was taken out and cutinto pieces of 5 cm×5 cm and then put on agar cultural media, culturedfor 48 hours at 32° C. There were three colonies of colon bacilli on anagar cultural medium.

The result shows that the adsorbent of Working Example 13 is excellentin adsorptive power of colon bacilli and possesses a germicidal power ofadhered colon bacilli. The result also shows that the adsorbent ofmicroorganisms of Working Example 13 does not cause the system to clogand is capable of efficiently removing microorganisms in an atmosphere.

WORKING EXAMPLE 14

An adsorptive resin was obtained as described in Working Example 13.

Table 10 shows the contents of the above operation.

On the other hand, a fiber web made of separate-type fiber (0.3 denierafter separation) comprising compositions of polyester and nylon wastreated by water entanglement, and a nonwoven fabric of 90 g/m² and 0.5mm in thickness was obtained.

An adsorbent of microorganisms (adsorptive resin is 1.5 wt % of nonwovenfabric) was obtained by impregnating an ethanol solution of saidadsorptive resin and lithium chloride in the proportion of 100:150 intothe nonwoven fabric and drying at 75° C.

Said adsorbent (10 cm×120 cm) was pleated and made into a filter unit of10 cm×10 cm. A spray box of colon bacilli was installed on theupperstream side of the filter unit and a suction pump with a membranousfilter of 0.45 μm was installed on the downstream side.

Removing said filter unit as a blank, a diluted isotonic sodium chloridesolution containing solon bacilli was sprayed and an atmospherecontaining colon bacilli was sucked by the pump at a speed of 10 l/minfor 2 minutes.

After having moisturized the absorbent well by sending through sterilemoist air for 5 minutes, a diluted isotonic sodium chloride solutioncontaining colon bacilli was sprayed and an atmosphere containing colonbacilli was sucked by the pump at a speed of 10 l/min. for 2 minutes.

Impregnating a liquid medium into the membranous filter as a blank andthe membranous filter after going through said filter unit, the filterswere cultured for 24 hours at 32° C. and the number of colonies of colonbacilli was counted each. The removal rates of colon bacilli taken fromthe counting are shown in Table 11.

                  TABLE 10                                                        ______________________________________                                                                         2,2'-                                                                         azobisiso-                                   chemical 4-vinyl          benzyl buty-   ethyl                                compound pyridine stylene bromide                                                                              ronitrile                                                                             alcohol                              ______________________________________                                        abbreviation                                                                           4VP      ST      BzBr   AIBN    EtOH                                 ratio of 1        2.5                                                         monomer                                                                       mol %    28.57    71.43   28.57  0.2                                          (monomer =                                                                    100)                                                                          molecular                                                                              105.14   104.15  171.04 164.21  46.07                                weight                                                                        (g/mol)                                                                       purity   100      100     98     98      99                                   (%)                                                                           calculative                                                                            1,000    2,500   1,000  7                                            annexing                                                                      (m mol)                                                                       annexing 105.14   260.38  174.53 1.17    662.42                               amount                                                                        (g)                                                                           ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                                     Working Example 14                                               ______________________________________                                        Number of Colonies                                                                           285                                                            (blank)                                                                       Number of Colonies                                                                           63                                                             after going through                                                           Adsorbent                                                                     Removal Rate   78                                                             (%)                                                                           ______________________________________                                    

As is apparent from Table 11, the adsorbent of microorganisms of WorkingExample 14 is excellent in adsorptive power and is capable ofeffectively adsorbing microorganisms in an atmosphere.

The "agar plate culture method" which is used throughout the workingexamples is approximately as follows.

It is the most popular method to count the number of livingmicroorganisms. However, in case of a lowly contaminated sample, namelyless than the range 10 to 30/g(ml), the method is not utilized as thecount is not accurate.

(1) Subjects:

Internal solidifier (dispersion solidifier), X-ray contrast medium,local liquid medicine, hydrophile ointment, liquid medicine containinginsoluble matter, general internal liquid medicine, other water solublesolidifiers, etc.

(2) Amount:

Generally 1 g (ml), if possible, 10 g (ml).

(3) Medium:

Use either the following (1) or (2).

(1) SCD agar: in case a sample does not contain any antisepticsubstances or germicides.

(2) SCDLP agar: in case a sample contains an antiseptic substance or agermicide.

(4) Container:

Petri dishes of 8.5 to 9.0 cm.

(5) Culturing:

The supernatant liquid of an undiluted or diluted solution of aregulated sample is divided into Petri dishes 1 ml each. Usually two ormore Petri dishes are used for a single sample. Each dish of the liquidis poured in media (15 to 20 ml each) which have been melted by heat andkept at 48° C. to 50° C., and mixed well by revolving the media. Afterhaving them solidfied horizontally, the surfaces are dried well avoidinginfection with microorganisms. These media are held upside down for atleast 72 hours at 30° C. to 32° C. and the number of colonies iscounted.

When the concentration of a sample is high, or a sample is alreadyturbid before the culturing, the amount should be 0.5 ml and four ormore Petri dishes should be used.

(6) Judgment:

Count the number of colonies in each Petri dish, calculate the averagenumber in each diluted solution in each step, calculate the average interms of the amount of the sample and the rate of dilution, and get thenumber of living microorganisms for every gram (ml) of the subject. Thecounting method of colonies is as follows.

1) In case counting the number of microorganisms after 72 hours ofculturing is not possible due to the generation of proliferation ofcolonies, use the number of microorganisms taken after 48 hours instead.

b 2) In principle, use the number of microorganisms taken for a sampleat a stage when the number of colonies in each Petri dish is in therange 20 to 200 (30 to 300 is also acceptable) or in the vicinity.

3 ) When the number of colonies in each Petri dish is less than 10 atany stage of the dilution, count the number of colonies of the leastdiluted one, and do not record the average of the actual numbers. Forinstance, record as <1.0×10² in the case of a diluted liquid to tentimes. It is useful to keep the records of colors, shapes, sizes, etc.of the generated colonies to later determine the types ofmicroorganisms.

A adsorptive resin as described above possesses an excellent adsorptivepower and is soluble in an organic solvent. Therefore, it can beimpregnated into other base materials as a solution or can be coated.Also the use of the adsorptive resin is not limited as in the case ofcross-linked polyvinyl pyridinium halide which can be treated only as asolid matter. The surface area per unit weight of said adsorptive resinof microorganisms, which is an absolutely important factor for aneffective adsorbent, can be freely obtained by selecting base materialsand a desired adsorbent of microorganism can be also obtained.Adsorptive resins of the present invention are insoluble in water andtherefore do not contaminate the environment.

The adsorbent of microorganism, of the present invention when used inwater treatment, effectively adsorbs microorganisms and the adsorptivepower does not deteriorate with time, hardly causing clogging of thesystem. When the adsorbent is used for a biosensor or a bioreactor, itcan efficiently collect organisms in the air or water in a living andactive state. Especially when a three-dimensional base material such asa nonwoven fabric is used, the surface area to contact with reactivesubstances as well as solutions to be analyzed becomes large, enablingorganic catalytic functions to function satisfactorily.

Therefore, the adsorbent of microorganisms is very suitable for abioreactor or a biosensor that utilizes enzyme contained inmicroorganisms, produces reactive products and sorts substances.

The adsorbent of microorganisms of the present invention can effectivelyremove microorganisms by sterilizing, with a germicide, microorganismscollected by an adsorptive resin or by preventing the microorganismsfrom propagating. Especially when a porous material is used as a basematerial, an excellent adsorptive power can be expected, for the contactarea of the base material is big and the porous material itself gives afiltering effect. The collected microorganisms are either sterilized bya germicide or prevented from propagating. Therefore, the clogginghazard of the system is prevented and the life of the system iselongated.

As described, the adsorbent of microorganisms of the present inventioncan effectively remove microorganisms in the air or water and it issuitable for a water treatment substance or for a mask or an airfilter.

The adsorbent of microorganisms of the present invention does notcontaminate the environments since the germicide is insoluble in water.

The adsorbent of microorganisms of the present invention can collectmicroorganisms effectively even in the air as it can retain anadsorptive power helped by a moistener. When a porous material is usedas a base material, the surface area becomes large, enabling theadsorbent to possess a high adsorptive power of microorganisms, andtroubles such as clogging can be effectively prevented.

The adsorbent of microorganisms can collect microorganisms in the aireffectively and is suitable for a carrier of a biosensor or abioreactor.

The adsorbent of microorganisms of the present invention preventsmicroorganisms collected by the adsorbent from propagating, eliminatingtrouble such as clogging, and elongates the life of a mask or anairfilter.

Therefore the adsorbent of microorganisms is suitable for a mask or anairfilter.

In accordance with the method of the present invention for makingadsorbents of microorganisms, excellent adsorbents of microorganisms canbe easily made as the adsorptive resins of the present invention aresoluble in an organic solvent, enabling impregnation, spraying andcoating of the resins to the surface of a base material.

What is claimed is:
 1. A resin for adsorbing microorganisms comprising avinyl copolymer of the following formula ##STR3## wherein, R₁ is abenzyl group, an alkyl group of 4 to 16 carbon atoms or apentafluorophenylmethyl group, R₂ is a hydrogen atom or an alkyl groupof 1 to 3 carbon atoms, X is a halogen atom, and Y is a hydrogen atom,an alkyl group of 1 to 3 carbon atoms, a benzyl group, an ether group, acarboxyl group, a carboxylic acid ester group or an aryl group, saidvinyl copolymer being insoluble in water and soluble in organic solvent.2. An adsorbent for microorganisms comprising a vinyl copolymer of thefollowing formula and existing on the surface of a substrate, ##STR4##wherein, R₁ is a benzyl group, an alkyl group of 4 to 16 carbon atoms ora pentafluorophenylmethyl group, R₂ is a hydrogen atom or an alkyl groupof 1 to 3 carbon atoms, X is a halogen atom, and Y is a hydrogen atom,an alkyl group of 1 to 3 carbon atoms, a benzyl group, an ether group, acarboxyl group, a carboxylic acid ester group or an aryl group, saidvinyl copolymer being insoluble in water and soluble in organic solvent.3. The adsorbent according to claim 2, wherein said substrate is aporous material.
 4. The adsorbent according to claim 3, wherein saidsubstrate is a non-woven fabric.
 5. The adsorbent according to claim 2,wherein said vinyl copolymer is existing on the surface of a substratewith a germicidal agent.
 6. The adsorbent according to claim 3, whereinsaid vinyl copolymer is existing on the surface of a substrate with agermicidal agent.
 7. The adsorbent according to claim 4, wherein saidvinyl copolymer is existing on the surface of a substrate with agermicidal agent.
 8. The adsorbent according to claim 5, wherein saidgermicidal agent is water insoluble.
 9. The adsorbent according to claim6, wherein said germicidal agent is water insoluble.
 10. The adsorbentaccording to claim 7, wherein said germicidal agent is water insoluble.11. The adsorbent according to claim 2 wherein said vinyl copolymer isexisting on the surface of a substrate with a moisture adsorbent. 12.The adsorbent according to claim 3 wherein said vinyl copolymer isexisting on the surface of a substrate with a moisture absorbent. 13.The adsorbent according to claim 4 wherein said vinyl copolymer isexisting on the surface of a substrate with a moisture absorbent.